This invention resides in the field of clinical assays indicative of biological conditions, and is of interest in the technology of binding assays for analytes in biological fluids for purposes of diagnosis, monitoring, or other clinical functions.
Since the initial disclosure of radioimmunoassays in 1961, a wide variety of in vitro assays using affinity-type binding have been developed. Variations include the type of binding (for example, specific vs. non-specific, and immunological vs. non-immunological), the type of detection (including the use of labels such as enzyme labels, radioactive labels, fluorescent labels, and chemiluminescent labels), methods of detecting whether or not binding has occurred (including methods in which bound species are separated from unbound species and methods that do not include such separation), and various other aspects of the assay procedure. The technology is currently used for the detection and quantitation of countless species, and serves as an analytical tool in the detection and monitoring of many physiological conditions and functions and the diagnosis and treatment of many diseases.
Of particular interest are assays that determine thyroid hormone levels. Accurate assessment of thyroid hormone levels is critical to determining thyroid function status. The free fraction of thyroid hormone in serum is only about 0.03% for thyroxin (T4) (I) and about 0.3% for 3,5,3xe2x80x2-triiodothyronine (T3) (II). Many methods have been developed for measuring serum thyroid hormone content, including labeled analogue-radioimmunoassay, ultrafiltration, column fractionation, and direct equilibrium dialysis followed by radioimmunoassay (Kapstein et al., J. Clin. Endocrin. Metab. 52: 1073-1079 (1981)). 
Although radioimmunoassays are of use in determining thyroid hormones in serum, there has been an effort by both industry and academia to replace radioisotopes with non-isotopic labels in clinical assays. Thyroid hormone assays that use non-radioactive analogues of thyroid hormones have been developed. For example, assays relying on fluorescent thyroid hormone analogues (Khosravi et al., Clin. Chem. 39: 256-262 (1993); Piran et al., J. Immunol. Methods 133: 207-214 (1990); Adamczyk et al., U.S. Pat. No. 5,691,456)) and chemiluminescent thyroid hormone analogues (Law et al., J. Bioluminescence and Chemiluminescence 4: 88-98 (1989)) to generate a signal are known in the art.
Numerous thyronine derivatives that include a detectable label within their framework are known in the art. The labeled thyronine derivatives are slight modifications of the phenyl ether nucleus of thyronine. For example, Wissman et al. (U.S. Pat. No. 4,820,860) disclose thyronine derivatives in which an iodine substituent on the thyronine nucleus is replaced by a moiety that includes a detectable label. Danielson et al. (U.S. Pat. No. 5,527,709) disclose an immunoassay using a label that includes a thyronine nucleus tethered to a detectable labeling group by means of a linking chain. U.S. Pat. No. 4,741,897, to Andrews et al. discloses compounds in which the thyronine nucleus is modified by attaching a linking group having a iodinatable aryl or heteroaryl group bound thereto. Other references disclosing compounds having a derivatized thyronine nucleus include, Feinberg (U.S. Pat. No. 4,711,855); Adamczyk et al., (U.S. Pat. No. 5,691,456, and Bioconjugate Chem. 5: 459-462 (1994)). To produce each of the agents disclosed in the above-enumerated references, the thyronine nucleus must be obtained and its integrity maintained during subsequent synthetic elaboration to produce the thyronine derivatives.
Fluorescent and chemiluminescent assays for thyroid hormones typically measure only serum T4 content. Assays in which both T3 and T4 are measured are important to the appropriate diagnosis and treatment of thyroid disease. Thus, efforts have been directed to developing assays that measure both serum T3 and T4 levels. With the exception of certain radioisotopic methods (see, Chopra, Thyroid 8: 249-257 (1998)), assays that are also capable of measuring T3 content require a separate assay mixture and a separate probe for measuring T3. Each additional assay probe, mixture and step adds additional expense and requires additional effort by technical staff. Thus, a non-radioactive immunoassay that detected both T3 and T4 levels in serum using a single assay mixture would represent a significant advance in the field. An even further advance would result from the use of detectable assay probes that were structurally simple and could be easily and inexpensively prepared.
It has now been discovered that structurally simple fluorescent analogues of thyroid hormones in which the thyronine nucleus is replaced by a monocyclic phenol moiety cross-react with antibodies raised against the thyroid hormones T3 and T4, and are useful in immunoassays for detecting T3 and T4. The new fluorescent analogues are easily prepared from readily available, inexpensive starting materials. The presence of a highly fluorescent label (e.g., phycobiliprotein) within the structure of the compounds of the invention allows their detection at low concentrations
Thus, in a first aspect, the present invention provides T3 and T4 thyroid hormone analogues that are labeled with a highly fluorescent phycobiliprotein. The fluorescent hormone analogues are easily prepared and characterized. Furthermore, the fluorescent hormone analogues interact with antibodies raised against T3 and T4 and compete with these hormones for the antibody hapten binding site.
The compounds of the invention have structures according to Formulae III-VI, below: 
in which PBP is a phycobiliprotein.
In a second aspect, this invention provides an immunoassay for individually determining T3 and T4 thyroid hormones in a sample using one or more compound of the invention. The immunoassay includes, forming an assay mixture by contacting the biological sample with a plurality of microparticles and a fluorescent thyroid hormone analogue of the invention. The microparticles are classifiable into a first population and a second population, which are distinguishable from each other by an automated detection means. The first population of microparticles has an anti-T3 antibody coupled to the particles. The second population of microparticles has an anti-T4 antibody coupled to the particles. In one embodiment, the fluorescent thyroid hormone analogue has a structure according to Formula V. Alternatively, the fluorescent analogue is a mixture of compounds according to Formula III and one of compound IV or VI. The particles are then recovered from the assay mixture and the fluorescence from the first and second populations is detected, thereby determining the amount of T3 thyroid hormone and T4 thyroid hormone in the biological sample.
These and other features and advantages of the invention will be more readily understood by the description that follows.